Rapamycin and its analogues bind tightly to the FK506-binding protein (FKBP) family of immunophilins (Cao & Konsolaki, 2011; Gerard et al., 2011). The FKBP family consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions (Kang et al., 2008). All FKBPs contain a domain with prolyl cis/trans isomerase (PPlase) activity. Binding of rapamycin or analogues to this domain inhibits their PPlase activity while mediating immune suppression through inhibition of mTOR. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration (Cao & Konsolaki, 2011; Gerard et al., 2011; Bove et al., 2011; Kang et al., 2008). Rapamycin is a nM inhibitor of the PPlase activity of several neurophilins including FKBP12 and FKBP52, and binding to these proteins has been shown to contribute to their neuroprotective effects (Ruan et al, 2008). FKBP52 binds Tau, and Tau protein overexpression is linked to inhibition of neurite outgrowth and neuroprotection (Chambraud et al., 2010). FKBP52 controls chemotropic guidance of neural growth cones via regulation of TRPC1 channel opening (Shim et al., 2009). These data provide a link for the neurite outgrowth promoting, axonal regeneration and neuroprotective effects observed for FKBP52 knockdown/inhibition. FKBP12 has been proposed many times as the major mediator of the neuroprotective effects of immunophilins, for example FK506 protection against oxygen-glucose deprivation induced damage was not present when an anti-FKBP12 antibody was added (Labrande et al., 2006), expression of FKBP12 is increased in the brain of patients with Parkinson's Disease, Alzheimer's disease and some forms of dementia (Avramut et al., 2002). It has also been implicated as the most potent enhancer of α-synuclein aggregation (Gerard et al., 2010, Deleersnijder et al., 2011).
Macrophage infectivity potentiators (MIPs) are close homologues of human FKBPs and have been shown to be important for virulence in some bacteria, such as Burkholderia sp., and inhibition of these MIPs by rapamycin has been shown (Norville et al, 2011). Other bacteria containing MIPs include Neisseria sp. (Leuzzi et al. 2005), Legionella sp., Pseudomonas sp., Xanthomonas sp. (Zang et al., 2007), Acetinobacter sp., Chlamydia sp., Salmonella sp. and Klebsiella sp. They are also important in certain parasites, including Trypanosoma sp. (Moro et al. 1995). Inhibition of bacterial virulence factors is potentially an antimicrobial strategy that is nondestructive to the bacteria. It has been proposed that virulence inhibitors could constitute a new class of antibiotics (Travis et al. 2000). By affecting virulence mechanisms without challenging bacterial viability directly, these antibiotic agents would potentially place little or no pressure on the bacterial cell for the emergence of resistant strains (Crunkhorn 2008). However, rapamycin also inhibits the mTOR pathway, leading to potent immunosuppressive activity. This is obviously an undesirable side-effect for an FKBP- or MIP-inhibiting compound.
One object of the invention is therefore to identify rapamycin analogues which are potent FKBP (including but not limited to FKBP12, FKBP38, FKBP51 and FKBP52 or FKBP11, FKBP14, FKBP1, FKBP8, FKBP4, FKBP5, FKBP10, FKBP9, FKBP6, FKBP7, FKBP10, FKBP3 or FKBP2) and/or MIP inhibitors, but with reduced or modulated mTOR inhibitory activity, such as shown by reduced activity in a t-cell proliferation assay (e.g. a PLP t-cell proliferation assay or MLR assay) or an increased ratio of this assay to FKBP inhibition (e.g. PLP IC50/FKBP IC50) as compared to rapamycin or known rapamycin analogues.
In particular, reduction of mTOR inhibition with maintenance or increase in inhibition of bacterial MIPs could enable broad spectrum virulence inhibition, with activity against bacterial strains resistant to many current antibiotics, where there is a dire need for new therapies (especially Klebsiella sp., Acetinobacter sp., Neisseria sp., Legionella sp. and Pseudomonas sp.).